Absence of Connective Tissue Growth Factor (CTGF/CCN2) Alters Epidermal Growth Factor (EGF) Signaling in Palatogenesis
Joseph T. Tarr, B.S.1, Timothy G. Visser, B.S.1, Steven N. Popoff, Ph.D.1, James P. Bradley, M.D.2.
1Lewis Katz School of Medicine at Temple University, Philadelphia, PA, USA, 2Temple University Hospital, Philadelphia, PA, USA.
PURPOSE: Nonsyndromic cleft palate is a common craniofacial birth defect with an incidence of 1 in 700 live births. Recent studies have shown that connective tissue growth factor (CTGF/CCN2) has acted as a necessary downstream mediator of TGF-β-dependent mesenchymal stem cell proliferation in palatogenesis. In our laboratory, in CTGF/CCN2 knockout (KO) mice, we identified numerous craniofacial defects such as failure of secondary palate formation. We investigated this novel cleft (CTGF/CCN2 KO) model’s anatomy, histology, and cellular function compared to the wild-type (WT).
METHODS: Palates were isolated from CTGF/CCN2 KO and WT E14.5 day mice and mRNA expression examined by qRT-PCR for alterations in known palatogenesis signaling pathways. Mesenchymal cells were isolated from CTGF/CCN2 WT and KO palates and used for protein expression analysis of genes that were found to have significantly altered mRNA expression. Mesenchymal cells were used to assess for responsiveness to recombinant CTGF/CCN2 and recombinant EGF.
RESULTS: mRNA isolated from palates demonstrated decreased EGFR expression in CTGF/CCN2 KO embryos. Mesenchymal cells isolated from CTGF/CCN2 KO palate demonstrates decreased protein expression of EGFR consistent with the observed mRNA decrease. Recombinant CTGF/CCN2 produced no effect on cellular proliferation. Recombinant EGF was found to increase proliferation in WT cells but unable to stimulate proliferation in CTGF/CCN2 KO cells.
CONCLUSION: Defects in proliferation significantly contribute to the clefting phenotype observed in the CTGF/CCN2 mouse model. CTGF/CCN2 interacts with several pathways involved in proliferation. Evaluation of our model has revealed abnormalities in EGF signaling in the knockout that lead to decreased proliferation. Further examination of these pathways will contribute to our understanding of the cleft etiology and may elucidate novel targets for clinical management of this birth defect.
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